Defects in the transforming growth factor beta (TGF-beta) signal transduction pathway are well-known in pancreatic cancer;the most well-established alteration is mutation of the Smad4, originally known as deleted in pancreatic cancer 4 (Dpc-4), which occurs in approximately 50% of human pancreatic cancers examined. TGF-beta binding to its transmembrane receptors leads to phosphorylation of Smad2 and Smad3, which form heterotrimeric complexes with Smad4 to inhibit growth of normal pancreatic epithelial cells. In pancreatic cancer cells, TGF-beta-induced growth inhibition is prevented either by loss of Smad4 or by inhibiting Smad-dependent growth arrest. Heider and colleagues recently reported that Smad- dependent growth arrest in pancreatic cancer cells is inhibited by overexpression of the protein Ski, a protein known to bind directly to Smads to inhibit signaling. Nine of 11 human pancreatic cancer specimens examined overexpressed Ski, which was not expressed in 9 normal pancreatic specimens. siRNA knockdown of Ski in Panc-1 cells restored TGF-beta-induced p21 expression and growth inhibition in culture and in a xenograft model (Heider et al., Annals of Surgery 246: 61-68, 2007). Over-expression of Ski also has been implicated in the cancer phenotype of esophageal squamous cell carcinoma, melanoma and cervical carcinoma. We propose a pilot project to test the hypothesis that pharmacological disruption of the Ski-Smad interaction will restore TGF-beta signaling in Ski-overexpressing pancreatic cancer cells. We hope that these studies might lead to a novel therapeutic approach for treating some pancreatic cancers. We have identified small constrained peptides, called peptide aptamers, and one small molecule ligand that inhibits Smad3-Ski binding. In Specific Aim 1, we propose to optimize our initial aptamer reagents for disrupting Smad-Ski interactions in pancreatic cancer cells over-expressing Ski and to define the Ski protein binding "hot spots" on Smads. In Specific Aim 2, we propose to target the Ski-binding hot spots for discovery of additional small molecules ligands through screening of available chemical libraries. In Specific Aim 3, our initial active compound and additional drug-like chemicals from the Aim 2 screens will be optimized for their potency and selectivity in restoring TGF-beta growth inhibition in Ski-overexpressing human pancreatic cancer cells. PUBLIC HEALTH RELEVANCE: Most pancreatic cancer patients treated with current therapies survive for only 3 to 6 months;the overall 5- year survival rate for this disease is <5%. We propose pre-clinical studies to evaluate a new therapeutic target in pancreatic cancer cells, the Ski-Smad protein interaction. We will use peptide aptamers and drug- like chemicals to disrupt Ski-Smad, restore an important growth inhibitory pathway in the pancreatic cancer cells, and provide proof-of-principle that Ski-Smad is a valid, druggable target for new drug development to aid pancreatic cancer patients.